Bispecific sigma-1 receptor antagonism and mu-opioid receptor partial agonism: WLB-73502, an analgesic with improved efficacy and safety profile compared to strong opioids

Opioids are the most effective painkillers, but their benefit-risk balance often hinder their therapeutic use. WLB-73502 is a dual, bispecific compound that binds sigma-1 (S1R) and mu-opioid (MOR) receptors. WLB-73502 is an antagonist at the S1R. It behaved as a partial MOR agonist at the G-protein pathway and produced no/unsignificant β-arrestin-2 recruitment, thus demonstrating low intrinsic efficacy on MOR at both signalling pathways. Despite its partial MOR agonism, WLB-73502 exerted full antinociceptive efficacy, with potency superior to morphine and similar to oxycodone against nociceptive, inflammatory and osteoarthritis pain, and superior to both morphine and oxycodone against neuropathic pain. WLB-73502 crosses the blood-brain barrier and binds brain S1R and MOR to an extent consistent with its antinociceptive effect. Contrary to morphine and oxycodone, tolerance to its antinociceptive effect did not develop after repeated 4-week administration. Also, contrary to opioid comparators, WLB-73502 did not inhibit gastrointestinal transit or respiratory function in rats at doses inducing full efficacy, and it was devoid of proemetic effect (retching and vomiting) in ferrets at potentially effective doses. WLB-73502 benefits from its bivalent S1R antagonist and partial MOR agonist nature to provide an improved antinociceptive and safety profile respect to strong opioid therapy.

Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis / 药物分析学报

Morphine is a frequently used analgesic that activates the mu-opioid receptor(MOR),which has prominent side effects of tolerance.Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance,currently,there is no effective therapy to treat morphine tolerance.In the current study,we aimed to develop a monoclonal antibody(mAb)precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms.We successfully prepared a mAb targeting MOR,named 3A5C7,by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization,and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation.Treatment of two cell lines,HEK293T and SH-SY5Y,with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2(GRK2)/β-arrestin2-dependent mechanism,as demonstrated by immunofluorescence staining,flow cytometry,Western blotting,coimmunoprecipitation,and small interfering ribonucleic acid(siRNA)-based knock-down.This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR.We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid.Western blot,enzyme-linked immunosorbent assays,and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase,the in vitro biomarker of morphine tolerance,via the GRK2/β-arrestin2 pathway.Furthermore,in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alle-viated morphine tolerance in mice,and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence.Finally,intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/β-arrestin2 pathway.Collectively,our study provided a therapeutic mAb,3A5C7,targeting MOR to treat morphine tolerance,mediated by enhancing morphine-induced MOR endocytosis.The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.

Mu-Opioid Receptors Expressed in Glutamatergic Neurons are Essential for Morphine Withdrawal / 神经科学通报·英文版

Although opioids still remain the most powerful pain-killers, the chronic use of opioid analgesics is largely limited by their numerous side-effects, including opioid dependence. However, the mechanism underlying this dependence is largely unknown. In this study, we used the withdrawal symptoms precipitated by naloxone to characterize opioid dependence in mice. We determined the functional role of mu-opioid receptors (MORs) expressed in different subpopulations of neurons in the development of morphine withdrawal. We found that conditional deletion of MORs from glutamatergic neurons expressing vesicular glutamate transporter 2 (Vglut2) largely eliminated the naloxone-precipitated withdrawal symptoms. In contrast, conditional deletion of MORs expressed in GABAergic neurons had a limited effect on morphine withdrawal. Consistently, mice with MORs deleted from Vglut2 glutamatergic neurons also showed no morphine-induced locomotor hyperactivity. Furthermore, morphine withdrawal and morphine-induced hyperactivity were not significantly affected by conditional knockout of MORs from dorsal spinal neurons. Taken together, our data indicate that the development of morphine withdrawal is largely mediated by MORs expressed in Vglut2 glutamatergic neurons.

Mu-Opioid Receptors Expressed in Glutamatergic Neurons are Essential for Morphine Withdrawal / 神经科学通报·英文版

Although opioids still remain the most powerful pain-killers, the chronic use of opioid analgesics is largely limited by their numerous side-effects, including opioid dependence. However, the mechanism underlying this dependence is largely unknown. In this study, we used the withdrawal symptoms precipitated by naloxone to characterize opioid dependence in mice. We determined the functional role of mu-opioid receptors (MORs) expressed in different subpopulations of neurons in the development of morphine withdrawal. We found that conditional deletion of MORs from glutamatergic neurons expressing vesicular glutamate transporter 2 (Vglut2) largely eliminated the naloxone-precipitated withdrawal symptoms. In contrast, conditional deletion of MORs expressed in GABAergic neurons had a limited effect on morphine withdrawal. Consistently, mice with MORs deleted from Vglut2 glutamatergic neurons also showed no morphine-induced locomotor hyperactivity. Furthermore, morphine withdrawal and morphine-induced hyperactivity were not significantly affected by conditional knockout of MORs from dorsal spinal neurons. Taken together, our data indicate that the development of morphine withdrawal is largely mediated by MORs expressed in Vglut2 glutamatergic neurons.

Mu-opioid receptors in the paraventricular nucleus regulate ejaculatory behaviors via the sympathetic nerve system in male rats / 中华男科学杂志

Objective@#To explore the effects of the mu-opioid receptor (MOR) in the paraventricular nucleus (PVN) on the ejaculatory behaviors of male rats and its potential mechanisms.@*METHODS@#Male SD rats with normal ejaculation ability were mated with female ones in hormone-induced estrus. After bilateral PVN microinjection of D-Ala-2-Me-Phe-4-Gly-ol enkephalin (DAGO) or D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) with an inserted catheter, the male animals were observed for mount latency (ML), mount frequency (MF), intromission latency (IL), intromission frequency (IF), ejaculation latency (EL), ejaculation frequency (EF), post-ejaculation interval (PEI), and intromission ratio (IR). The lumbar sympathetic nerve activity (LSNA) of the rats was recorded using the PowerLab data acquisition hardware device, and the levels of norepinephrine (NE) in the peripheral plasma were measured by ELISA following microinjection of saline or different doses of DAGO or CTAP.@*RESULTS@#Neither CTAP nor DGAO significantly affected the ML of the male rats (P > 0.05). DGAO remarkably increased IF (P < 0.01) and MF (P < 0.01), prolonged IL (P < 0.01), EL (P < 0.01) and PEI (P < 0.01), and reduced EF (P <0.01) and IR (P < 0.05). On the contrary, CTAP markedly decreased IF (P < 0.01) and MF (P < 0.01), shortened IL (P < 0.01), EL (P < 0.01) and PFI (P < 0.01), and elevated EF (P < 0.01) and IR (P < 0.01). Additionally, DAGO decreased LSNA in a dose-dependent manner and reduced the NE level in the peripheral plasma. CTAP, however, not only offset the effects of DAGO on LSNA, but also significantly increased LSNA.@*CONCLUSIONS@#MOR in PVN inhibits ejaculatory behaviors in male rats by weakening LSNA, which has provided some theoretical evidence for the use of highly selective opioids in the treatment of premature ejaculation.

Anti-nociceptive effect of patchouli alcohol: Involving attenuation of cyclooxygenase 2 and modulation of mu-opioid receptor / 中国结合医学杂志

OBJECTIVE@#To explore the anti-nociceptive effect of patchouli alcohol (PA), the essential oil isolated from Pogostemon cablin (Blanco) Bent, and determine the mechanism in molecular levels.@*METHODS@#The acetic acid-induced writhing test and formalin-induced plantar injection test in mice were employed to confirm the effect in vivo. Intracellular calcium ion was imaged to verify PA on mu-opioid receptor (MOR). Cyclooxygenase 2 (COX2) and MOR of mouse brain were expressed for determination of PA's target. Cellular experiments were carried out to find out COX2 and MOR expression induced by PA.@*RESULTS@#PA significantly reduced latency period of visceral pain and writhing induced by acetic acid saline solution (P<0.01) and allodynia after intra-plantar formalin (P<0.01) in mice. PA also up-regulated COX2 mRNA and protein (P<0.05) with a down-regulation of MOR (P<0.05) both in in vivo and in vitro experiments, which devote to the analgesic effect of PA. A decrease in the intracellular calcium level (P<0.05) induced by PA may play an important role in its anti-nociceptive effect. PA showed the characters of enhancing the MOR expression and reducing the intracellular calcium ion similar to opioid effect.@*CONCLUSIONS@#Both COX2 and MOR are involved in the mechanism of PA's anti-nociceptive effect, and the up-regulation of the receptor expression and the inhibition of intracellular calcium are a new perspective to PA's effect on MOR.

Can oliceridine (TRV130), an ideal novel µ receptor G protein pathway selective (µ-GPS) modulator, provide analgesia without opioid-related adverse reactions?

All drugs have both favorable therapeutic and untoward adverse effects. Conventional opioid analgesics possess both analgesia and adverse reactions, such as nausea, vomiting, and respiratory depression. The opioid ligand binds to µ opioid receptor and non-selectively activates two intracellular signaling pathways: the G protein pathway induce analgesia, while the β-arrestin pathway is responsible for the opioid-related adverse reactions. An ideal opioid should activate the G protein pathway while deactivating the β-arrestin pathway. Oliceridine (TRV130) has a novel characteristic mechanism on the action of the µ receptor G protein pathway selective (µ-GPS) modulation. Even though adverse reactions (ADRs) are significantly attenuated, while the analgesic effect is augmented, the some residual ADRs persist. Consequently, a G protein biased µ opioid ligand, oliceridine, improves the therapeutic index owing to increased analgesia with decreased adverse events. This review article provides a brief history, mechanism of action, pharmacokinetics, pharmacodynamics, and ADRs of oliceridine.

The involvement of mu opioid receptorin peripheral regulation of chronic inflammatory pain induced by CFAin rats / 中国比较医学杂志

Objective To investigate the change of mu opioid receptor ( MOR) in dorsal root ganglion ( DRG) in rat chronic inflammatory pain model and the effect of MOR agonist and antagonis tintraplantarly ( i.pl.) injected on pain threshold, so as to determine the role of peripheral MOR in chron in inflammatory pain .Methods Chronicin flammatory pain model was established by i .pl.injection of CFA in rats.The expression of MOR in DRG was detected by immunohistochemistry .Pain threshold before and after i .pl.injection of MOR agonist and antagonist was measured by radiant heat method .Results Rats suffered from an intraplantar injection of CFA developed chronic inflammatory pain , and the painthreshold still reduced on 18 day after CFA injection compared to that in the normal group . Immunohistochemistry staining revealed that compared with the normal group , the expression of MOR in DRG of CFA rats was increased ( P<0.01 ) .After the paw dorsal surface injection of MOR agonist , the pain threshold of CFA rats was increased, while that of normal rats exhibited no significant change .After the paw dorsal surface injection of MOR antagonist, the pain threshold of CFA rats was reduced , while that of normal rats had no significant change .Conclusion Under chronic inflammatory pain condition , DRG MOR expressionis enhanced , which participates in the regulation of chronic inflammatory pain , and may contribute to the prevention of further more serious pain .

Melanocortin-4 receptor expression in the cuneiform nucleus is involved in modulation of opioidergic signaling / 华中科技大学学报（医学）（英德文版）

Substantial evidence has suggested that deep brain stimulation of the cuneiform nucleus has become a remarkable treatment option for intractable pain, but the possible mechanism is poorly understood. Using a melanocortin-4 receptor (MC4R)-green fluorescent protein (GFP) reporter knockin mouse, we showed that a large number of MC4R-GFP-positive neurons were expressed in the cuneiform nucleus. Immunofluorescence revealed that approximately 40%-50% of MC4R-GFP-positive neurons expressed mu opioid receptors, indicating that they were opioidergic signaling. Our findings support the hypothesis that MC4R expression in the cuneiform nucleus is involved in the modulation of opioidergic signaling.

Expression of μ-opioid receptor in synovium tissue in acute inflamed knee joint of dogs / 第二军医大学学报

Objective: To observe the changes of mu-opioid receptors (MOR) expression in acute inflamed knee joint synovium tissue of dogs, so as to discuss the feasibility of using peripheral local opioid for analgesia in acute inflammation. Methods: Knee joint synovium tissues were taken from 9 dogs with acute arthritis and 8 dogs with normal knee joints. The expression of MOR protein and mRNA was examined by immunohistochemistry and real-time quantitative PCR. Results: The expression of MOR mRNA in the acute inflamed group was significantly higher than that in the normal control group ([34.40±5.48] % vs [16.54±8.03] %, P<0.05). Immunohistochemical result showed more positive staining of MOR particles and stronger signal in the acute arthritis group than in normal control group. The immunohistochemical index of MOR positive cells in the acute arthritis was significantly higher than that in normal control group ([323 175.00±92 614.94] vs [175 444.10±75 149.06], P< 0.05). Conclusion: MOR exists in the knee joint synovium tissue of dogs, and acute inflammation can enhance the expression of MOR.

The Mechanical Antiallodynic Effect of intrathecal Morphine and R-Phenylisopropyl-Adenosine in Rats with Spinal Nerve Ligation / 대한마취과학회지

BACKGORUND: A nerve ligation injury may produce a pain syndrome including mechanical allodynia. Usually the antiallodynic effect of morphine is diminished in a neuropathic rat model. However, in a previous study, spinal morphine was found to have an antiallodynic effect in a neuropathic rat model. Therefore, the present study was performed to observe the mechanical antiallodynic effects of spinal morphine and R-PiA, and to investigate the relationship between the two. METHODS: Male SD rats were prepared by tightly ligating the left L5 and L6 spinal nerve and by implanting a lumbar intrathecal catheter. in study 1, each of the 5 groups (morphine at 3 or 10mug, adenosine A1 receptor agonist (R-PiA) at 3 or 10mug, or saline) were administered intrathecally to examine changes in the mechanical allodynia threshold. in study 2, selective adenosine A1 receptor antagonist (DPCPX 10mug) was administered to investigate the reversal of the mechanical antiallodynic effect in the 4 treated groups. in study 3, we observed the pretreatment effect of DPCPX 10mug. The mechanical allodynic thresholds for left hindpaw withdrawal to von Frey hairs were assessed and converted to %MPE. RESULTS: in study 1, the mechanical allodynic threshold was significantly increased in a similar pattern by intrathecal morphine (3, 10mug) and R-PiA (3, 10mug) (P<0.05). in study 2, the allodynic threshold of morphine was insignificantly decreased by intrathecal DPCPX pretreatment. The mechanical allodynic threshold of R-PiA 3mug was decreased by intrathecal DPCPX (P<0.05). in study 3, the antiallodynic effect of morphine was not influenced by DPCPX pretreatment. CONCLUSiONS: intrathecal morphine and R-PiA produced the antiallodynic effect. The antiallodynic effect of morphine was slightly decreased by DPCPX 10mug. Therefore, it was suggested that the antiallodynic effect of morphine might be, at least in part, mediated by adenosine in a rat model of spinal nerve ligation.

Correlated Study of Heroin Dependence and Mu Opioid Receptor Gene / 昆明医科大学学报

Objective To detect relation between heroin dependence and mu opioid receptor gene.Methods Genotype and allele frequencies of polymorphisms of mu opioid receptor gene was examined in 313 heroin-dependence subjects and 214 normal controls.Results No difference in genotype and allele frequencies of polymorphism of mu opioid receptor gene were observed between heroin-dependent subjects and normal controls(?~2=3.73,P=0.16 and.?~2=0.76,P=0.38).Conclusion The results suggested that polymorphism of mu opioid receptor gene was not associated with heroin dependence.